Scallop S1 In order to visualize the conformational changes occurring in the myosin head during ATP hydrolysis, we are determining the structures of the native scallop myosin head fragment (S1) (produced by proteolysis) complexed in the active site with various nucleotide analogs. The current view is that different positions of the lever arm will be found. Thus far, a 2.5[unreadable] data set collected at CHESS allowed us to visualize the ADP-bound form of the molecule.This structure represents the highest resolution obtained to date for a myosin S1 fragment. We have also obtained a 4.2[unreadable] data set and determined the structure of the nucleotide-free state of scallop myosin S1 which corresponds to the last step of the contractile cycle. For the first time, we have been able to describe an ~35 degree tilting movement of the lever arm by comparing two states of the same myosin isoform. Moreover, we have been able to establish at the atomic level the nature of the conformational changes occurring in the motor domain (Houdusse et al., in preparation). A 3.8[unreadable] native data set of scallop S1 complexed with a MgADP.Pi analog (MgADP.vanadate) has also been collected. Structure determination of these crystals should lead to the characterization of a third conformation for the myosin head, that of the pre-power stroke . Vertebrate Smooth Muscle Myosin Head Fragments In parallel with the studies on invertebrate myosin S1 prepared by proteolysis, we have obtained crystals of several subfragment-nucleotide complexes of expressed smooth muscle myosin, a low velocity but high force vertebrate muscle myosin. Using data collected at CHESS, the crystal structures of an expressed vertebrate smooth muscle myosin motor domain (MD) and a motor domain-essential light chain (ELC) complex (MDE) with a transition state analog (MgADP.AlF4-) in the active site were determined to 2.9[unreadable] and 3.5[unreadable] resolution, respectively. The MDE structure with an ATP analog (MgADP.BeFx) was determined to 3.6[unreadable] resolution. In all three structures, the converter domain in the C-terminal region is rotated /70 from that in skeletal subfragment 1 (S1), although the presence of the ELC affects the precise position of the converter. A comparison of the lever arm positions in MDE-AlF4- and in skeletal S1 shows that a potential displacement of /13 nm can be achieved during the power stroke. The MDE-BeFx and MDE-AlF4-structures are almost identical, consistent with the fact that they both bind weakly to actin. These results imply that MgATP binding, and not hydrolysis, primes the lever arm for the power stroke (Dominguez et al., submitted). Non-Muscle Myosins Some members of the myosin superfamily are regulated by direct binding of calcium on the calmodulin subunits in their lever arm. We have grown crystals of a lever arm fragment of myosin V containing two calmodulins in the absence of calcium. A native data set to 2.9[unreadable] resolution as well as Pt and Au derivative data sets to about 3.5[unreadable] resolution have been collected at CHESS. We expect to determine this structure by MIR methods in the near future. The results will allow us to see how calmodulin can bind to its target on myosin under these conditions. These results will provide the first structure of the lever arm of an unconventional myosin, and are the starting point for understanding regulation in this system.